Original article
Bacteriobilia and fungibilia are associated with outcome in patients with endoscopic treatment of biliary complications after liver transplantation
Authors
Institutions
submitted 19. January 2012 accepted after revision 2. July 2013
Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1344713 Endoscopy 2013; 45: 890–896 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0013-726X Corresponding author D. N. Gotthardt, MD Department of Gastroenterology, Toxicology, and Infectious Diseases University Hospital of Heidelberg, Im Neuenheimer Feld 410 69120 Heidelberg, Germany Fax: +49-6221-565687 [email protected]
Daniel N Gotthardt1, *, Karl Heinz Weiss1, *, Christian Rupp1, Konrad Bode2, Isabella Eckerle3, Gerda Rudolph1, Janine Bergemann1, Petra Kloeters-Plachky1, Fadi Chahoud1, Markus W Büchler4, Peter Schemmer4, Wolfgang Stremmel1, Peter Sauer1 1 Department of Gastroenterology, Toxicology, and Infectious Diseases, University Hospital of Heidelberg, Germany 2 Department of Medical Microbiology and Hygiene, University Hospital of Heidelberg, Germany 3 Section of Clinical Tropical Medicine, University Hospital of Heidelberg, Germany 4 Department of Surgery, University Hospital of Heidelberg, Germany
Background and study aims: To determine the importance of bacteriobilia and fungibilia in patients with endoscopic treatment of biliary complications after orthotopic liver transplantation (OLT). Patients and methods: In a prospective study at a tertiary center, 213 patients underwent 857 endoscopic retrograde cholangiographies (ERCs) after OLT. Findings at first ERC were: anastomotic stricture in 24.4 %, nonanastomotic stricture in 18.3 %, leakage in 11.3 %, and gallstones in 4.7 %. Results: Bile samples from first ERC showed Gram-positive bacterial isolates in 102/180 (57 %) and Gram-negative in 44/180 (24 %). Main species were Enterococcus spp. (40 %; 72/180) and Escherichia coli (10 %; 18 /180). Enteric bacteria (present in 47 %) and Candida spp. (present in 18 %) were both associated with clinical signs of cholangitis, but not with laboratory signs of inflammation. Multiresistant strains (present in12 % of samples) showed no association with clinical or laboratory
parameters. Detection of microbiological isolates was independent of endoscopic findings and treatment. In patients with successful endoscopic intervention, the actuarial survival free of retransplantation was significantly lower in those with detection of enteric bacteria, being 51.8 months (95 % confidence interval [CI] 42.9 – 60.6) vs. 62.9 months (95 %CI 59.1 – 66.7); P = 0.025). Fungibilia was associated with significantly lower actuarial retransplantation-free survival, independently of successful endoscopic treatment (mean 35.1 months [95 %CI 23.5 – 46.7] vs. 53.1 months [(95 %CI 48.0 – 58.2]; P = 0.019). Conclusions: Bacteriobilia and fungibilia can frequently be detected by routine microbiological sampling in patients after OLT. Regular bile sampling is recommended since the presence of difficult-to-treat multiresistant strains is unpredictable. Survival is affected by this altered biliary microbiological environment after OLT.
Introduction
tervention has previously been the treatment of choice for all these situations, but, except for the last one, these can now be resolved by endoscopic means in most cases. Diffuse intrahepatic strictures offer no effective treatment options and regularly lead to the need for retransplantation. Endoscopic treatment options include sphincterotomy, rigid or balloon dilation, placement of one or more plastic stents, and removal of stones/debris or other obstructive material [5]. In non-OLT patients, biliary obstruction can be caused by, for example, choledocholithiasis, or benign or malignant strictures, and these situations are often complicated by biliary infection with symptoms ranging from mild cholangitis to sepsis with organ failure [6, 7]. In post-OLT patients with biliary complications, this infection is aggravated by immunosuppression. In addition, cholangitis without obvious complications of the biliary tract can complicate the course after OLT [8, 9].
!
Orthotopic liver transplantation (OLT) provides the only curative treatment option with excellent long-term results in patients with decompensated cirrhosis of the liver or acute liver failure. Biliary complications are an important cause of morbidity in these patients and occur in up to 10 % – 25 % of all OLT patients; the biliary reconstruction is often called the “Achilles’ heel” of OLT [1 – 4]. The three most frequent complications are strictures of the biliary tract, bile leaks (mostly at the site of the anastomosis), and biliary casts and stones. Strictures can occur at the site of anastomosis or at any other part of the biliary tract of the donor (nonanastomotic strictures). Nonanastomotic strictures are often categorized into strictures of extrahepatic donor ducts, hilar strictures, and diffuse intrahepatic strictures. Surgical re-in* Both authors contributed equally.
Gotthardt Daniel N et al. Bacteriobilia and fungibilia and endoscopic treatment of complications after liver transplantation … Endoscopy 2013; 45: 890–896
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890
Original article
Table 1 Baseline characteristics of 213 patients undergoing endoscopic retrograde cholangioscopy (ERC) for biliary complications following orthotopic liver transplant. Patients, n
213
ERCs, n
857
Age, mean (SD), years
53.5 (10.3)
Liver disease etiology, n (%) Cirrhosis, alcoholic
70 (33 %)
Cirrhosis, viral
63 (30 %)
Hepatocellular carcinoma
41 (19 %)
Other
39 (18 %)
891
with patients under conscious sedation with midazolam, propofol, and/or short-acting opiates. Many patients were on an antibiotic regimen before the initiation of ERC treatment because of suspected or proven cholangitis. The standard regimen in our institution is intravenous mezlocillin, or, in the case of known allergy to penicillins, ciprofloxacin was used. Patients not receiving antibiotic treatment who needed intervention received peri-interventional antibiotic prophylaxis (mezlocillin or ciprofloxacin). Candida infection was treated with fluconazole by mouth or intravenously. The baseline charac" Table 1. teristics of the patients are shown in ●
Laboratory parameters before ERC, mean (SD) 3.3 (3.7)
ALT, IU/L
158 (220)
AST, IU/L
106 (127)
AP, IU/L
392 (341)
gGT, IU/L
693 (632)
CRP, mg/L
31.0 (44.5)
WBC, /nL
7.4 (4.7)
SD, standard deviation; ERC, endoscopic retrograde cholangiography; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; gGT, gamma-glutamyltransferase; CRP, C-reactive protein; WBC, white blood cells
Biliary obstruction is often complicated by cholangitis, which can range from mild to life-threatening. Endoscopic treatment to enable biliary drainage and the use of antibiotics are both mainstays of treatment for this condition [10 – 12]. There is no agreement on the optimal initial antibiotic regimen [13], and limited information is available regarding the antibiotic susceptibility profile of the pathogens isolated from bile samples[14, 15]. Data on bacteriobilia or fungibilia, the presence of bacteria or fungi in bile, are scarce in OLT patients, although some are available for other diseases, for example in patients with primary sclerosing cholangitis (PSC) and gallstone disease [7, 16 – 18]. If data are available, the focus is on the microbiological flora, but correlation with clinical data is lacking, especially any survival analysis [19]. The clinical significance of these conditions and their influence on outcome in patients after OLT still need to be elucidated. In this prospective study we analyzed bacteriobilia and fungibilia in patients after OLT. We sought to identify risk factors, association with endoscopic treatment, clinical relevance, and impact on outcome in these patients.
Patients and methods !
Patients and endoscopic interventions This prospective study was conducted at a tertiary center, the University Hospital of Heidelberg, Germany, from December 2006 to March 2012. Patients’ follow-up data until June 2012 was acquired. Informed consent was provided by each patient, and the study was conducted in accordance with the Declaration of Helsinki and was approved by the local ethics committee. During the study period, 857 endoscopic retrograde cholangiography (ERC) procedures were undertaken in 213 patients with biliary complications after OLT. The ERCs were carried out using a therapeutic duodenoscope (TJF160 R or TJF160VR; Olympus, Tokyo, Japan), and selective cannulation of the common bile duct was done using a guidewire (Jagwire, 0.035-inch, Boston Scientific, Natick, Massachusetts, USA; or Visiglide 0.035-inch, Olympus) or a standard catheter for cases where there was a pre-existing sphincterotomy. All procedures were performed
Bile sampling and microbiological analysis Bile sampling and microbiological analysis was performed as described previously [17]. Briefly, bile samples were obtained after selective intubation before any therapeutic procedure was performed. When bile could not be aspirated directly after cannulation, a small amount of sterile saline (2 – 4 ml) was applied and aspiration was reattempted. Aliquots of all biliary samples were placed in a sterile glass tube containing medium for anaerobic and aerobic bacterial cultures (BD BBL Port-A-Cul; Becton, Dickinson and Co., Sparks, Maryland, USA). The material was delivered to the microbiology laboratory within 2 h of collection and cultured aerobically and anaerobically according to standard laboratory protocols. After endoscopic procedures all endoscopes were pre-cleaned at the point of use with detergent solution. After brushing of the endoscope channel, parts, connectors, and orifices, endoscopes were further reprocessed by automated high level disinfection according to the manufacturers’ instructions (Olympus EDT 3, Olympus; Korsolex Endo-cleaner, and Korsolex Endo-disinfectant, Bode Chemie, Hamburg, Germany). All reprocessing steps as well as the training of personnel involved conformed with guideline recommendations [20]. Cultures of endoscope surfaces, channels, and parts, and also of worktops were performed routinely by our local microbiology department. During the study period, no growth of germs was observed in any of the routinely performed cultures. The analysis of susceptibility to antibiotics commonly used for treatment included results for all isolates for which routine antibiotic susceptibility tests were performed. For further analysis, we categorized the samples into groups according to the bacterial and fungal findings: (i) sterile; (ii) low grade pathogens, consisting mainly of alpha- or beta-hemolytic streptococci and coagulase-negative staphylococcus; (iii) enteric bacteria, including Escherichia coli, Enterococcus spp., Klebsiella spp. and other Enterobacteriaceae; (iv) Candida, including all Candida species; and (v) multiresistant bacteria, which included all samples containing at least one multiresistant strain.
Study design, definitions and statistical analysis The study was designed to determine the incidence and spectrum of biliary infections and drug resistance and to assess the impact of these findings on outcome. The study was also designed to identify potential risk factors for biliary infection after OLT. Sustained clinical success of endoscopic treatment was defined by an intervention-free period for at least 3 months after the last endoscopic intervention. Assisted clinical success was defined as a recurrent necessity of endoscopic intervention to keep the patient free of cholestatic symptoms or clinically apparent cholangitis. Treatment failure of the initial endoscopic treatment
Gotthardt Daniel N et al. Bacteriobilia and fungibilia and endoscopic treatment of complications after liver transplantation … Endoscopy 2013; 45: 890–896
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Bilirubin, mg/dL
Original article
was defined as demonstration of continuous bile leakage after two attempts at treatment with plastic stents or persistence of a stricture and persistent cholestatic symptoms after 1 year after the start of treatment, despite multiple balloon dilations and/or use of plastic stents. Either surgical re-intervention or experimental endoscopic treatment with temporary placement of metal stents was performed. Recurrence of a biliary lesion was defined as diagnosis of a stricture or leak after a time interval of more than 3 months after the last intervention. In addition we recorded any newly developed biliary finding with a need for therapeutic intervention that was different from the initial lesion. Laboratory values for white blood cells (WBC), C-reactive protein (CRP), alkaline phosphatase (ALP), gamma-glutamyltransferase (gGT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and bilirubin were determined, and clinical signs of cholangitis, including fever, right upper quadrant pain, and jaundice were recorded before the ERCs were performed. These data are in line with published criteria for acute cholangitis [10]. Previous endoscopic intervention, laboratory parameters at baseline, clinical signs of cholangitis, and patient age were defined as potential risk factors for biliary infection. Continuous data were compared using the nonparametric Mann–Whitney U test. Frequency differences were compared using the chi-squared test or Fisher’s exact test where appropriate. The covariates that showed a high level of significance in the univariate analysis were analyzed further in a multivariate analysis using the binary logistic regression method. Actuarial retransplantation-free survival was estimated using a Kaplan–Meier product limit estimator. Differences between the actuarial estimates were tested using the log rank test. Cox regression was used to calculate the hazard ratio. Differences were considered significant when P < 0.05. Statistical analyses were performed using PASW Statistics 17.0 (SPSS, Chicago, Illinois, USA).
Table 2 Endoscopic findings in 213 patients at baseline (index endoscopic retrograde cholangioscopy [ERC] for biliary complications following orthotopic liver transplant.
n Indication for primary ERC Unexplained progressive cholestasis 1
91
42.7
Suspected stricture 1
72
33.8
Suspected leakage 1
38
17.8
Suspected gallstones 1
9
4.2
Cholangitis
3
1.4
49
23.0
Anastomotic stricture
52
24.4
Nonanastomotic stricture
39
18.3
Leakage
24
11.3
Previous endoscopic treatment (EST, balloon dilation, plastic stent, percutaneous drainage) Findings at primary ERC 2
Gallstones
10
4.7
Purulent cholangitis 3
12
5.6
115
53.9
Endoscopic treatment (balloon dilation, plastic stent, metal stent, stone removal, EST alone)
ERC, endoscopic retrograde cholangiography; EST, endoscopic sphincterotomy; 1 With or without signs of cholangitis 2 Where combined lesions were present, the clinically more prominent one was used 3 Purulent cholangitis was observed with or without other pathological findings
Table 3 Success of endoscopic treatment. Therapeutic procedures were carried out at the index ERC in 115 of the 213 patients with biliary complications after orthotopic liver transplant.
n/n
%
Sustained clinical success
60/115
52.2
Assisted clinical success
21/115
18.3
Combined (sustained or assisted) clinical success
81/115
70.4
Therapeutic procedure at first ERC (n = 115)
Success not evaluable Treatment failure
Results !
Endoscopic findings and treatment after liver transplantation Overall, 213 patients who had undergone liver transplantation were included in this prospective study. The underlying liver diseases had been chronic viral hepatitis (n = 63), alcoholic liver disease (n = 70), hepatocellular carcinoma (n = 41), or other indications (n = 39). The median time interval between transplantation and the occurrence of the biliary complication requiring the index ERC was 3.0 months (0 – 252 months). Indications for performing ERC included suspected stricture (n = 72), leakage (n = 38) or bile duct stones (n = 9). Out of the 213 patients, 21 presented with clinical signs of cholangitis; laboratory parameters of inflammation for the 213 patients were as follows: WBC mean (SD) 7.6 (5.0) /nL, CRP 31.0 (42.9) mg/L (upper limit of normal 5.0 mg/L). Three patients presented with clinical signs of severe cholangitis or sepsis, but in a substantial proportion of patients with progressive cholestasis (n = 91) neither ultrasound, magnetic resonance imaging (MRI), computed tomography (CT) scan, nor histology was able to provide a reliable diagnosis. Therefore in these patients ERC was performed primarily with a diagnostic intention. This led to definite diagnosis in 36 % of these patients. In 23 % (49/213) of patients a previous endoscopic biliary intervention was documented (endoscopic sphincterotomy [EST] 11.3 %, balloon dilation 6.6 %, plastic stent placement 3.8 %, other
%
Recurrent or newly developed biliary lesion (among all patients, n = 213)
7/115
6.1
27/115
23.5
21/213
9.8
ERC, endoscopic retrograde cholangioscopy
1.4 %). This intervention had been either performed before OLT or at a different endoscopy unit. During the ERC, the most common finding was anastomotic stricture, followed by nonanastomotic or diffuse intrahepatic strictures, leakage or gallstones. In 115 patients (53.9 %) one or more therapeutic endoscopic pro" Table 2). The sustained clinical succedures were performed (● cess rate in these patients was 52 % (60/115) and the assisted clinical success rate 18 % (21/115). Thus, an overall clinical success after the endoscopic interventions was achieved in 70 % of patients. Treatment failure occurred in 23.5 % (27/115). Recurrence of the biliary complication or a newly developed biliary lesion " Taduring follow up was observed in 10 % (21/213) of patients (● ble 3).
Detection rates for bacteria and fungi in bile Among all the ERC examinations of the 213 patients at inclusion into the prospective study, bile cultures were not performed in 33 cases because of technical or organizational failure. In total, 102/180 (57 %) of patients had Gram-positive and 44/180 " Table 4). (24 %) had Gram-negative bacteria detectable in bile (● Enterococcus species were predominant (72/180 samples; 40 %). Escherichia. coli and Klebsiella spp. were detected in 10 % (18/180) and 4 % (7 /180) of the samples, respectively. Alpha- and beta-he-
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892
Table 4 Microbiological isolates from bile specimens at first endoscopic retrograde cholangiography (ERC).
Organism
n
%1
Bacteria At least one Gram-positive:
102
56.7
Enterococcus spp.
72
40.0
Streptococci, alpha- and beta-hemolytic
37
20.6
Staphylococcus spp.
23
12.8
Other
9
5.0
44
24.4
Escherichia coli
18
10.0
Klebsiella spp.
7
3.9
Enterobacter cloacae
3
1.7
Neisseria spp.
3
1.7
Citrobacter spp.
2
1.1
Pseudomonas aeruginosa
2
1.1
18
10.0
1
0.6
At least one Gram-negative:
Other Multiresistant bacteria Vancomycin-resistant enterococci (VRE) Multiresistant Enterococcus faecium, not VRE
12
6.7
Extended spectrum beta lactamase (ESBL)
2
1.1
Other multiresistant strains
7
3.9
Fungi Candida albicans
1
24
15.6
C. tropicalis
2
1.3
C. glabrata
7
4.5
Candida spp.
3
1.9
Sterile
54
(30.0)
No information available
33
(15.5)
Percentage is based on either the total number of ERCs for which microbiological analysis was available (n = 180) or the total number of ERCs where bile was in addition tested for fungi (n = 154)
Table 5
Microbiological spectrum of bile samples.
%1
n
1
Sterile
54
30 %
Low grade pathogens
57
32 %
Enteric
85
47 %
Candida
28
18 %
Any multiresistant strain
21
12 %
As percentage of ERCs for which microbiological information was available (154 for Candida; 180 for the rest).
Bile sample microbiology
Laboratory signs of cholangitis
molytic streptococci were detected in 21 % of the samples and coagulase-negative staphylococci in 13 %. The Gram-negative species were Enterobacter cloacae (2 %), Citrobacter spp. (1 %), and Pseudomonas aeruginosa (1 %). Multiresistant bacteria included the following: 1 % vancomycin-resistant Enterococcus (VRE) faecalis, 1 % E. coli with extended spectrum beta-lactamase (ESBL) resistance, and 7 % Enterococcus faecium strains that were only sensitive to " Table 4). vancomycin, linezolid, or other reserve antibiotics (● We also analyzed the bile samples for fungi. A total of 16 % showed Candida albicans; however, other Candida species were " Table 4 and ● " Table 5). Some Candida detected at low rates (● species, including C. glabrata and C. tropicalis, were detected at low frequencies, while still other fungal organisms, such as Aspergillus fumigatus, were not detected. Summarizing the results of analysis resulted in the following: 54/ 180 (30 %) sterile bile cultures; 57/180 (32 %) samples with low grade pathogens; 85/180 (47 %) with at least one enteric bacterial strain; 28/180 (18 %) samples with Candida. At least one multiresistant strain was detected in 21 /180 (12 %) of the samples. These " Table 5. data are summarized in ●
Risk factors for detection of microbiological isolates according to univariate and multivariate analyses For analysis of bacterial isolates we included 180/213 patients in the analysis, while for patients with fungibilia we included 154/ 213 patients, since in 33 patients microbiological data were not available and for 26 patients data on bacteribilia, but not fungibilia were available. Neither bacteriobilia arising from enteric bacteria nor detection of Candida was associated with increased serum levels of CRP " Table 6). WBC was not associated with the presence of bacter(● ia or fungi. The clinical signs of cholangitis were recorded before ERC treatment and were much more frequent in the groups with enteric bacteribilia and fungibilia (P < 0.05 for all comparisons). There was no association with either of the low grade pathogen groups (data not shown). As with the clinical signs of cholangitis, patient age and previous endoscopic intervention were associated with the detection of " Table 7). Lower levels found in liver function enteric bacteria (● test (LFT) results were significantly associated with the detection
Clinical signs of inflammation Values are n (%)
WBC, /nL
CRP, mg/L
– ve
+ ve
Sterile
7.8 (4.8)
27.7 (43.1)
51 (96 %)
2 (4 %)
Not sterile
7.2 (4.7)
32.4 (45.1)
111 (87 %)
16 (13 %)
Univariate analysis
P = 0.135
P = 0.583
P = 0.101
Multivariate analysis 1
—
—
—
Enteric– ve
7.5 (4.4)
27.3 (42.2)
91 (96 %)
3 (4 %)
Enteric + ve
7.1 (5.1)
35.1 (47.3)
71 (84 %)
15 (16 %)
Univariate analysis
P = 0.361
P = 0.313
P = 0.003
Multivariate analysis 1
—
—
P = 0.051
Candida – ve
7.4 (5.0)
31.2 (46.7)
119 (94 %)
7 (6 %)
Candida + ve
7.4 (4.5)
32.8 (45.9)
22 (79 %)
6 (21 %)
Univariate analysis
P = 0.868
P = 0.434
Multiresistant – ve
7.5 (4.8)
29.9 (44.4)
134 (90 %)
15 (10 %)
Multiresistant + ve
7.9 (5.9)
36.7 (37.5)
18 (86 %)
Univariate analysis
P = 0.799
P = 0.157
Table 6 Clinical and laboratory signs of cholangitis. Univariate and multivariate analysis. Parameter values are means (standard deviation [SD]), with units as shown.
P = 0.006 3 (14 %) P = 0.470
WBC, white blood cell count; CRP, C-reactive protein; – ve, absent; + ve, present WBC and CRP were analyzed using the Mann – Whitney U test. Significance of differences was calculated using the chi-squared or Fisher’s exact test where applicable. 1 Parameters with P < 0.1 in univariate analysis were included in a multivariate analysis. The P values from this analysis are reported here.
Gotthardt Daniel N et al. Bacteriobilia and fungibilia and endoscopic treatment of complications after liver transplantation … Endoscopy 2013; 45: 890–896
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Original article
Original article
Table 7 Patient age, liver function tests and previous endoscopic intervention associated with detection of bacteria and fungi at first ERC. Parameter values are means (SD), with units as shown.
Bile sample
Age, years
AST, IU/L
ALT, IU/L
ALP, IU/L
gGT, IU/L
microbiology
Bilirubin,
No previous inter-
Previous endoscopic
mg/dL
vention, n(%)
intervention, n (%)
Not sterile
54.2 (10.7)
101 (137)
140 (211)
358 (313)
628 (601)
3.1 (3.8)
87 (68 %)
Sterile
51.9 (9.1)
117 (96)
203 (235)
482 (395)
854 (684)
3.7 (3.5)
47 (89 %)
40 (32 %)
Univariate analysis
P = 0.034
P = 0.007
P = 0.001
P = 0.019
P = 0.024
P = 0.058
Multivariate analysis 1
P = 0.684
P = 0.395
P = 0.158
P = 0.175
P = 0.890
P = 0.782
Enteric– ve
52.4 (9.3)
115 (108)
183 (217)
428 (380)
786 (645)
3.3 (3.3)
83 (87 %)
12 (13 %)
Enteric + ve
55.1 (11.1)
97 (146)
132 (224)
345 (285)
582 (583)
3.3 (4.2)
51 (60 %)
34 (40 %)
Univariate analysis
P = 0.005
P = 0.004
P = 0.001
P = 0.073
P = 0.012
P = 0.088
Multivariate analysis
P = 0.563
P = 0.675
P = 0.202
P = 0.415
P = 0.625
P = 0.444
Candida – ve
53.8 (10.6)
99 (112)
142 (181)
395 (335)
709 (621)
3.3 (3.8)
99 (79 %)
27 (21 %)
Candida + ve
52.4 (10.0)
153 (194)
215 (317)
444 (412)
616 (693)
3.5 (4.0)
18 (64 %)
10 (36 %)
Univariate analysis
P = 0.529
P = 0.823
P = 0.908
P = 0.745
P = 0.211
P = 0.914
Multiresistant – ve
53.8 (10.5)
103 (104)
152 (193)
402 (352)
688 (620)
3.4 (3.9)
111 (75 %)
Multiresistant + ve
52.0 (10.4)
150 (246)
238 (384)
381 (287)
837 (711)
3.5 (3.7)
17 (81 %)
Univariate analysis
P = 0.649
P = 0.370
P = 0.771
P = 0.966
P = 0.354
P = 0.837
6 (11 %) P = 0.005 P = 0.007
P < 0.001 P = 0.001
P = 0.110 38 (25 %) 4 (19 %) P = 0.601
ERC, endoscopic retrograde cholangioscopy; SD, standard deviation; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; gGT, gamma-glutamyltransferase. The Mann – Whitney U test was used to analyze for laboratory values; the chi-squared test was used to compare intervention. 1 Parameters with P < 0.1 in univariate analysis were included in a multivariate analysis. The P values from this analysis are reported here.
" Taof enteric bacteria and the detection of any isolate in bile (● ble 7). Parameters that were associated with a P value < 0.1 were all analyzed using a logistic regression model. For detection of sterile bile and for the detection of enteric bacteria in bile only a previous intervention remained significant (P < 0.01). We assessed whether the presence of bacteribilia and/or fungibilia was associated with endoscopic findings in patients after liver transplantation. There was no significant association with the frequency of endoscopic findings nor with the frequency of sustained endoscopic therapeutic success with the detection rate of microbiological isolates (P > 0.05, chi-square test; data not shown).
Association of bacteriobilia and fungibilia with retransplantation free survival We assessed whether the presence of bacteriobilia and/or fungibilia was associated with outcome in patients undergoing endoscopic treatment after liver transplantation. Patients with detection of enteric bacteria at first ERC had no difference in regard to retransplantation-free survival compared with patients without enteric bacteria, if endoscopic treatment success was not considered. In patients who were successfully treated by endoscopic intervention, the actuarial retransplantation-free survival was significantly shorter when enteric bacteria were detected: in those positive for enteric bacteria it was 51.8 months (95 % confidence interval [CI] 42.9 – 60.6), vs. 62.9 months (95 %CI 59.1 – 66.7) in those negative for enteric bacteria, " Fig. 1 a), with a hazard ratio of 7.620. with P = 0.025 (● Fungibilia was associated with significantly shorter actuarial retransplantation-free survival, independently of the success of endoscopic treatment. Patients with detection of Candida had a mean actuarial retransplantation-free survival of 35.1 months (95 %CI 23.5 – 46.7) whereas those without Candida had a mean " Fig. 1 b), with a hazard raof 53.1 months (95 %CI 48.0 – 58.2) (● tio of 2.590. When patients with a previous endoscopic intervention were excluded the results were as follows. Patients who were successfully treated, showed an actuarial retransplantation-free survival of
62.4 months (95 %CI 57.6 – 67.2) if no enteric bacteria were detected vs. 51.4 months (95 %CI 39.9 – 62.8) if enteric bacteria were present (P = 0.057). Patients without a previous intervention and without Candida had a mean actuarial retransplantation-free survival after first ERC of 50.4 months (95 %CI 44.4 – 56.4); whereas patients with Candida showed a mean of 27.5 months (95 %CI 16.3 – 38.7) (P = 0.049.
Discussion !
Cholangitis is a serious complication of biliary obstruction that requires biliary drainage and antibiotic treatment [10 – 12, 21]. Following transplantation, infectious complications are a major cause of morbidity and mortality [22]. Immunosuppression is one factor contributing to this, but in addition, in patients who have had liver transplantation the biliary tract is impaired in its physiological function to a varying extent. Therefore cholangitis can occur more easily. This leads to biliary tract infection as a major complication in these patients. Data on frequencies of bacteriobilia and cholangitis in patients after OLT are scarce, and were regularly obtained from patients with a T-tube drain in place during the first days after transplantation. Fungal species are emerging as pathogens that are detected frequently in the setting of healthcare-associated biliary infection. In agreement with previous reports [23, 24] we detected Candida species, mainly C. albicans in bile samples. We were able to confirm the high frequency of bacteriobilia and fungibilia that has been reported by Millonig et al. in a smaller previous study [19]. In our cohort, only 30 % of samples were sterile. These numbers are noteworthy, since bile is normally considered to be sterile. It is of interest that even at first ERC almost 50 % of samples showed enteric bacteria and 18 % showed Candida. One of the main findings of this study is the clear benefit of routine bile sampling during ERCs. Bile cultures can be used to identify bacterial strains and, more importantly, if performed routinely reveal a patient’s susceptibility profile. Blood cultures are far less sensitive for detecting this information and the inclusion of such data from bile sampling can lead to changes in the anti-
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894
1.0 enteric –ve 0.8
Cumulative survival
enteric +ve 0.6 P < 0.05 0.4
0.2
enterio-negative enterio-positive censored censored
0.0 0 a
20 40 Time from first ERC, months
60
1.0
Candida –ve
Cumulative survival
0.8
0.6 Candida +ve 0.4 P < 0.05 0.2
Candida-negative Candida-positive censored censored
0.0 0 b
20 40 Time from first ERC, months
60
Fig. 1 Bacteriobilia and fungibilia in liver transplantation patients: association with survival without further transplantation. The microbiological assessment was done at a first endoscopic retrograde cholangioscopy (ERC) performed because of biliary complications after orthotopic liver transplantation (OLT). All time periods are from the first ERC of the study. a Among patients who were successfully treated by endoscopic intervention, those with no enteric bacteria in their bile showed a significantly better actuarial survival free of retransplantation, with a mean 62.9 months (95 % confidence interval [CI] 59.1 – 66.7) vs. those with enteric bacteriobilia, with a mean 51.8 months (95 %CI 42.9 – 60.6); P = 0.025. b Patients without Candida present in their bile had a higher retransplantation-free survival, with a mean of 53.1 months (95 %CI 48.0 – 58.2) vs. patients with Candida with a mean of 35.1 months (95 %CI 23.5 – 46.7); P = 0.019. This finding was independent of success of endoscopic treatment.
biotic treatment [18]. This is especially important in patients who have had liver transplantation, because such patients are regularly hospitalized and treated for infectious complications of different kinds. Since we could not identify clear risk factors for the detection of multiresistant strains, we argue for the acquisition of bile samples during each ERC. Although bile cultures are more sensitive, blood cultures are still standard in patients with signs of infection. Usually acquisition of bile is possible without any difficulty. In cases when aspiration is cumbersome, the endoscopist needs to weigh the potential benefit of bile culture ver-
sus the potentially higher risk of the procedure and contamination of the sample because of prolonged acquisition time. In our cohort the predominance of Enterococcus spp. was even more obvious than had been anticipated, with 40 % of all samples being positive for any kind of Enterococci. This is in line with other data relating to intra-abdominal infection post-OLT [25] but it is higher compared with other reports from patients with obstructive jaundice [14, 15, 26], and is of special importance because Enterococcus spp. have been shown to be largely resistant to fluoroquinolones, especially ciprofloxacin [27]. Ciprofloxacin has been widely used as peri-interventional prophylaxis in ERC patients and in treatment of cholangitis. Our data would argue in favor of a broad spectrum penicillin or, in cases of any known multiresistant bacterial strain in bile and cholangitis, for even broader antimicrobial therapy such as carbapenems. On the basis of our data we cannot give a recommendation on the duration of antibiotic treatment. We found different Candida species in our analysis. No other fungal organism, e. g. Aspergillus spp., was identified. Therefore treatment with fluconazole should be sufficient, but the optimal duration of treatment has to be determined. Usually we treat for 3 – 4 weeks which is longer than treatment for bacteria. The clinical relevance of bacteriobilia and fungibilia has been questioned. We have demonstrated their clinical relevance by the significant association between clinical signs of cholangitis with bacteriobilia/fungibilia although numerous patients had bacteriobilia/fungibilia and no clinical sign of cholangitis. This might either reflect the impaired immunological response in the immunosuppressed patient, a subclinical course of cholangitis, or simply the altered biliary microenvironment of these patients after OLT. Although all patients received antibiotic treatment, eradication of the bacteriobilia was not achieved. So in post-OLT patients, the biliary tract can no longer be considered to be a sterile environment. This altered biliary microenvironment might be further challenged by changes in the biliary lipid composition leading to nonanastomotic stricture and graft failure, resembling biliary cirrhosis caused by reduced phospholipid content [28, 29]. We could not find a significant association with the endoscopic findings nor with the success rate of the endoscopic treatment. In a considerable proportion of patients (43 %) ERC was performed with a diagnostic purpose, because histological examination as well as imaging procedures were not able to clarify the progressive cholestasis. As a definitive diagnosis was thereby established in more than one third of patients, this approach appears appropriate. The influence on survival has not been addressed in former studies on OLT patients, but has revealed significant results in patients with PSC [30, 31]. The detection of Candida was associated with a markedly shorter retransplantation-free survival. Whether this effect is due to a chronic, damaging infection of the biliary tract or whether biliary tract candidiasis is only another marker of disease has yet to be determined. Furthermore we were able to show that the initial absence of enteric bacteria is associated with longer retransplantation-free survival in patients who respond to endoscopic therapy. This is noteworthy, because it shows that the altered microbiological environment of the biliary tract after liver transplantation contributes to outcome in these patients. We also confirmed the excellent results of endoscopic therapy in patients with biliary tract complications after liver transplantation [32 – 34]. Two points have to be kept in mind when interpreting our data. The first concerns the potential cross-contamination. As all routi-
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Original article
nely performed cultures of endoscopes, involved surfaces, and worktops were negative, cross-contamination from reprocessing and storage of endoscopes could be excluded; however crosscontamination during the upper gastrointestinal passage of the endoscope may have influenced the results. The other point is that many of our patients had received antibiotic treatment before bile sampling was performed. Since the exact timing of application of the antibiotics and bile sampling could not be retrieved, potential antimicrobiological activity in the bile might influence the microbiological assessment. In conclusion, in post-liver transplantation patients, bacteriobilia and fungibilia can frequently be detected by routine microbiological sampling, thereby allowing optimized, strain-specific antibiotic treatment. Both have clinical significance and we also identified significant risk factors for the detection of bacteria and Candida. Since we could not identify clear risk factors for the detection of multiresistant strains, we argue for acquisition of bile samples during each ERC. This would enable the physician to administer targeted strain-specific treatment. For the first time, we assessed survival in these patients on the basis of biliary tract microbiology and depending on their endoscopic treatment and the success rate of endoscopic treatment. We demonstrated a shorter retransplantation-free survival in patients with Candida or enteric bacteria present in their bile. The influence of this microbiologically altered biliary environment on chronic graft failure and its treatment options warrants further study. Competing interests: None
Acknowledgments !
We thank Tom Bruckner, Department of Medical Biometry and Informatics, for critical review of the statistic analyses. D.G. and K.H.W. were supported by the post-doctoral program of the Medical Faculty of the University of Heidelberg. D.G. was also supported by a grant from the Deutsche Forschungsgemeinschaft.
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10 Wada K, Takada T, Kawarada Y et al. Diagnostic criteria and severity assessment of acute cholangitis: Tokyo Guidelines. J Hepatobiliary Pancreat Surg 2007; 14: 52 – 58 11 Lai EC, Mok FP, Tan ES et al. Endoscopic biliary drainage for severe acute cholangitis. N Engl J Med 1992; 326: 1582 – 1586 12 Deviere J, Motte S, Dumonceau JM et al. Septicemia after endoscopic retrograde cholangiopancreatography. Endoscopy 1990; 22: 72 – 75 13 Lee JG. Diagnosis and management of acute cholangitis. Nat Rev Gastroenterol Hepatol 2009; 6: 533 – 541 14 Nomura T, Shirai Y, Hatakeyama K. Enterococcal bactibilia in patients with malignant biliary obstruction. Dig Dis Sci 2000; 45: 2183 – 2186 15 Rosch T, Triptrap A, Born P et al. Bacteriobilia in percutaneous transhepatic biliary drainage: occurrence over time and clinical sequelae. A prospective observational study. Scand J Gastroenterol 2003; 38: 1162 – 1168 16 Bjornsson ES, Kilander AF, Olsson RG. Bile duct bacterial isolates in primary sclerosing cholangitis and certain other forms of cholestasis –ve a study of bile cultures from ERCP. Hepatogastroenterology 2000; 47: 1504 – 1508 17 Rudolph G, Gotthardt D, Klöters-Plachky P et al. Influence of dominant bile duct stenoses and biliary infections on outcome in primary sclerosing cholangitis. J Hepatol 2009; 51: 149 – 155 18 Negm AA, Schott A, Vonberg RP et al. Routine bile collection for microbiological analysis during cholangiography and its impact on the management of cholangitis. Gastrointest Endosc 2010; 72: 284 – 291 Epub 2010 Jun 11 19 Millonig G, Buratti T, Graziadei IW et al. Bactobilia after liver transplantation: frequency and antibiotic susceptibility. Liver Transpl 2006; 12: 747 – 753 20 Petersen BT, Chennat J, Cohen J et al. Multisociety guideline on reprocessing flexible gastrointestinal endoscopes: 2011. Gastrointest Endosc 2011; 73: 1075 – 1084 21 Rerknimitr R, Fogel EL, Kalayci C et al. Microbiology of bile in patients with cholangitis or cholestasis with and without plastic biliary endoprosthesis. Gastrointest Endosc 2002; 56: 885 – 889 22 Fishman JA. Infection in solid-organ transplant recipients. N Engl J Med 2007; 357: 2601 – 2614 23 Ehrenstein BP, Salamon L, Linde HJ et al. Clinical determinants for the recovery of fungal and mezlocillin-resistant pathogens from bile specimens. Clin Infect Dis 2002; 34: 902 – 908 24 Lenz P, Conrad B, Kucharzik T et al. Prevalence, associations, and trends of biliary-tract candidiasis: a prospective observational study. Gastrointest Endosc 2009; 70: 480 – 487 25 Reid GE, Grim SA, Sankary H et al. Early intra-abdominal infections associated with orthotopic liver transplantation. Transplantation 2009; 87: 1706 – 1711 26 Jethwa P, Breuning E, Bhati C et al. The microbiological impact of preoperative biliary drainage on patients undergoing hepato-biliary-pancreatic (HPB) surgery. Aliment Pharmacol Ther 2007; 25: 1175 – 1180 27 Werner G, Fleige C, Ewert B et al. High-level ciprofloxacin resistance among hospital-adapted Enterococcus faecium (CC17). Int J Antimicrob Agents 2009 28 Buis CI, Geuken E, Visser DS et al. Altered bile composition after liver transplantation is associated with the development of nonanastomotic biliary strictures. J Hepatol 2009; 50: 69 – 79 29 Gotthardt D, Runz H, Keitel V et al. A mutation in the canalicular phospholipid transporter gene, ABCB4, is associated with cholestasis, ductopenia, and cirrhosis in adults. Hepatology 2008; 48: 1157 – 1166 30 Gotthardt D, Stiehl A. Endoscopic retrograde cholangiopancreatography in diagnosis and treatment of primary sclerosing cholangitis. Clin Liver Dis 2010; 14: 349 – 358 31 Gotthardt DN, Rudolph G, Kloters-Plachky P et al. Endoscopic dilation of dominant stenoses in primary sclerosing cholangitis: outcome after long-term treatment. Gastrointest Endosc 2010; 71: 527 – 534 32 Kulaksiz H, Weiss KH, Gotthardt D et al. Is stenting necessary after balloon dilation of post-transplantation biliary strictures? Results of a prospective comparative study Endoscopy 2008; 40: 746 – 751 33 Sauer P, Chahoud F, Gotthardt D et al. Temporary placement of fully covered self-expandable metal stents in biliary complications after liver transplantation. Endoscopy 2012; 44: 536 – 538 34 Balderramo D, Navasa M, Cardenas A. Current management of biliary complications after liver transplantation: emphasis on endoscopic therapy. Gastroenterol Hepatol 2011; 34: 107 – 115
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Bacteriobilia and fungibilia are associated with outcome in patients with endoscopic treatment of biliary complications after liver transplantation
Authors
Institutions
submitted 19. January 2012 accepted after revision 2. July 2013
Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1344713 Endoscopy 2013; 45: 890–896 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0013-726X Corresponding author D. N. Gotthardt, MD Department of Gastroenterology, Toxicology, and Infectious Diseases University Hospital of Heidelberg, Im Neuenheimer Feld 410 69120 Heidelberg, Germany Fax: +49-6221-565687 [email protected]
Daniel N Gotthardt1, *, Karl Heinz Weiss1, *, Christian Rupp1, Konrad Bode2, Isabella Eckerle3, Gerda Rudolph1, Janine Bergemann1, Petra Kloeters-Plachky1, Fadi Chahoud1, Markus W Büchler4, Peter Schemmer4, Wolfgang Stremmel1, Peter Sauer1 1 Department of Gastroenterology, Toxicology, and Infectious Diseases, University Hospital of Heidelberg, Germany 2 Department of Medical Microbiology and Hygiene, University Hospital of Heidelberg, Germany 3 Section of Clinical Tropical Medicine, University Hospital of Heidelberg, Germany 4 Department of Surgery, University Hospital of Heidelberg, Germany
Background and study aims: To determine the importance of bacteriobilia and fungibilia in patients with endoscopic treatment of biliary complications after orthotopic liver transplantation (OLT). Patients and methods: In a prospective study at a tertiary center, 213 patients underwent 857 endoscopic retrograde cholangiographies (ERCs) after OLT. Findings at first ERC were: anastomotic stricture in 24.4 %, nonanastomotic stricture in 18.3 %, leakage in 11.3 %, and gallstones in 4.7 %. Results: Bile samples from first ERC showed Gram-positive bacterial isolates in 102/180 (57 %) and Gram-negative in 44/180 (24 %). Main species were Enterococcus spp. (40 %; 72/180) and Escherichia coli (10 %; 18 /180). Enteric bacteria (present in 47 %) and Candida spp. (present in 18 %) were both associated with clinical signs of cholangitis, but not with laboratory signs of inflammation. Multiresistant strains (present in12 % of samples) showed no association with clinical or laboratory
parameters. Detection of microbiological isolates was independent of endoscopic findings and treatment. In patients with successful endoscopic intervention, the actuarial survival free of retransplantation was significantly lower in those with detection of enteric bacteria, being 51.8 months (95 % confidence interval [CI] 42.9 – 60.6) vs. 62.9 months (95 %CI 59.1 – 66.7); P = 0.025). Fungibilia was associated with significantly lower actuarial retransplantation-free survival, independently of successful endoscopic treatment (mean 35.1 months [95 %CI 23.5 – 46.7] vs. 53.1 months [(95 %CI 48.0 – 58.2]; P = 0.019). Conclusions: Bacteriobilia and fungibilia can frequently be detected by routine microbiological sampling in patients after OLT. Regular bile sampling is recommended since the presence of difficult-to-treat multiresistant strains is unpredictable. Survival is affected by this altered biliary microbiological environment after OLT.
Introduction
tervention has previously been the treatment of choice for all these situations, but, except for the last one, these can now be resolved by endoscopic means in most cases. Diffuse intrahepatic strictures offer no effective treatment options and regularly lead to the need for retransplantation. Endoscopic treatment options include sphincterotomy, rigid or balloon dilation, placement of one or more plastic stents, and removal of stones/debris or other obstructive material [5]. In non-OLT patients, biliary obstruction can be caused by, for example, choledocholithiasis, or benign or malignant strictures, and these situations are often complicated by biliary infection with symptoms ranging from mild cholangitis to sepsis with organ failure [6, 7]. In post-OLT patients with biliary complications, this infection is aggravated by immunosuppression. In addition, cholangitis without obvious complications of the biliary tract can complicate the course after OLT [8, 9].
!
Orthotopic liver transplantation (OLT) provides the only curative treatment option with excellent long-term results in patients with decompensated cirrhosis of the liver or acute liver failure. Biliary complications are an important cause of morbidity in these patients and occur in up to 10 % – 25 % of all OLT patients; the biliary reconstruction is often called the “Achilles’ heel” of OLT [1 – 4]. The three most frequent complications are strictures of the biliary tract, bile leaks (mostly at the site of the anastomosis), and biliary casts and stones. Strictures can occur at the site of anastomosis or at any other part of the biliary tract of the donor (nonanastomotic strictures). Nonanastomotic strictures are often categorized into strictures of extrahepatic donor ducts, hilar strictures, and diffuse intrahepatic strictures. Surgical re-in* Both authors contributed equally.
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Original article
Table 1 Baseline characteristics of 213 patients undergoing endoscopic retrograde cholangioscopy (ERC) for biliary complications following orthotopic liver transplant. Patients, n
213
ERCs, n
857
Age, mean (SD), years
53.5 (10.3)
Liver disease etiology, n (%) Cirrhosis, alcoholic
70 (33 %)
Cirrhosis, viral
63 (30 %)
Hepatocellular carcinoma
41 (19 %)
Other
39 (18 %)
891
with patients under conscious sedation with midazolam, propofol, and/or short-acting opiates. Many patients were on an antibiotic regimen before the initiation of ERC treatment because of suspected or proven cholangitis. The standard regimen in our institution is intravenous mezlocillin, or, in the case of known allergy to penicillins, ciprofloxacin was used. Patients not receiving antibiotic treatment who needed intervention received peri-interventional antibiotic prophylaxis (mezlocillin or ciprofloxacin). Candida infection was treated with fluconazole by mouth or intravenously. The baseline charac" Table 1. teristics of the patients are shown in ●
Laboratory parameters before ERC, mean (SD) 3.3 (3.7)
ALT, IU/L
158 (220)
AST, IU/L
106 (127)
AP, IU/L
392 (341)
gGT, IU/L
693 (632)
CRP, mg/L
31.0 (44.5)
WBC, /nL
7.4 (4.7)
SD, standard deviation; ERC, endoscopic retrograde cholangiography; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; gGT, gamma-glutamyltransferase; CRP, C-reactive protein; WBC, white blood cells
Biliary obstruction is often complicated by cholangitis, which can range from mild to life-threatening. Endoscopic treatment to enable biliary drainage and the use of antibiotics are both mainstays of treatment for this condition [10 – 12]. There is no agreement on the optimal initial antibiotic regimen [13], and limited information is available regarding the antibiotic susceptibility profile of the pathogens isolated from bile samples[14, 15]. Data on bacteriobilia or fungibilia, the presence of bacteria or fungi in bile, are scarce in OLT patients, although some are available for other diseases, for example in patients with primary sclerosing cholangitis (PSC) and gallstone disease [7, 16 – 18]. If data are available, the focus is on the microbiological flora, but correlation with clinical data is lacking, especially any survival analysis [19]. The clinical significance of these conditions and their influence on outcome in patients after OLT still need to be elucidated. In this prospective study we analyzed bacteriobilia and fungibilia in patients after OLT. We sought to identify risk factors, association with endoscopic treatment, clinical relevance, and impact on outcome in these patients.
Patients and methods !
Patients and endoscopic interventions This prospective study was conducted at a tertiary center, the University Hospital of Heidelberg, Germany, from December 2006 to March 2012. Patients’ follow-up data until June 2012 was acquired. Informed consent was provided by each patient, and the study was conducted in accordance with the Declaration of Helsinki and was approved by the local ethics committee. During the study period, 857 endoscopic retrograde cholangiography (ERC) procedures were undertaken in 213 patients with biliary complications after OLT. The ERCs were carried out using a therapeutic duodenoscope (TJF160 R or TJF160VR; Olympus, Tokyo, Japan), and selective cannulation of the common bile duct was done using a guidewire (Jagwire, 0.035-inch, Boston Scientific, Natick, Massachusetts, USA; or Visiglide 0.035-inch, Olympus) or a standard catheter for cases where there was a pre-existing sphincterotomy. All procedures were performed
Bile sampling and microbiological analysis Bile sampling and microbiological analysis was performed as described previously [17]. Briefly, bile samples were obtained after selective intubation before any therapeutic procedure was performed. When bile could not be aspirated directly after cannulation, a small amount of sterile saline (2 – 4 ml) was applied and aspiration was reattempted. Aliquots of all biliary samples were placed in a sterile glass tube containing medium for anaerobic and aerobic bacterial cultures (BD BBL Port-A-Cul; Becton, Dickinson and Co., Sparks, Maryland, USA). The material was delivered to the microbiology laboratory within 2 h of collection and cultured aerobically and anaerobically according to standard laboratory protocols. After endoscopic procedures all endoscopes were pre-cleaned at the point of use with detergent solution. After brushing of the endoscope channel, parts, connectors, and orifices, endoscopes were further reprocessed by automated high level disinfection according to the manufacturers’ instructions (Olympus EDT 3, Olympus; Korsolex Endo-cleaner, and Korsolex Endo-disinfectant, Bode Chemie, Hamburg, Germany). All reprocessing steps as well as the training of personnel involved conformed with guideline recommendations [20]. Cultures of endoscope surfaces, channels, and parts, and also of worktops were performed routinely by our local microbiology department. During the study period, no growth of germs was observed in any of the routinely performed cultures. The analysis of susceptibility to antibiotics commonly used for treatment included results for all isolates for which routine antibiotic susceptibility tests were performed. For further analysis, we categorized the samples into groups according to the bacterial and fungal findings: (i) sterile; (ii) low grade pathogens, consisting mainly of alpha- or beta-hemolytic streptococci and coagulase-negative staphylococcus; (iii) enteric bacteria, including Escherichia coli, Enterococcus spp., Klebsiella spp. and other Enterobacteriaceae; (iv) Candida, including all Candida species; and (v) multiresistant bacteria, which included all samples containing at least one multiresistant strain.
Study design, definitions and statistical analysis The study was designed to determine the incidence and spectrum of biliary infections and drug resistance and to assess the impact of these findings on outcome. The study was also designed to identify potential risk factors for biliary infection after OLT. Sustained clinical success of endoscopic treatment was defined by an intervention-free period for at least 3 months after the last endoscopic intervention. Assisted clinical success was defined as a recurrent necessity of endoscopic intervention to keep the patient free of cholestatic symptoms or clinically apparent cholangitis. Treatment failure of the initial endoscopic treatment
Gotthardt Daniel N et al. Bacteriobilia and fungibilia and endoscopic treatment of complications after liver transplantation … Endoscopy 2013; 45: 890–896
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Bilirubin, mg/dL
Original article
was defined as demonstration of continuous bile leakage after two attempts at treatment with plastic stents or persistence of a stricture and persistent cholestatic symptoms after 1 year after the start of treatment, despite multiple balloon dilations and/or use of plastic stents. Either surgical re-intervention or experimental endoscopic treatment with temporary placement of metal stents was performed. Recurrence of a biliary lesion was defined as diagnosis of a stricture or leak after a time interval of more than 3 months after the last intervention. In addition we recorded any newly developed biliary finding with a need for therapeutic intervention that was different from the initial lesion. Laboratory values for white blood cells (WBC), C-reactive protein (CRP), alkaline phosphatase (ALP), gamma-glutamyltransferase (gGT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and bilirubin were determined, and clinical signs of cholangitis, including fever, right upper quadrant pain, and jaundice were recorded before the ERCs were performed. These data are in line with published criteria for acute cholangitis [10]. Previous endoscopic intervention, laboratory parameters at baseline, clinical signs of cholangitis, and patient age were defined as potential risk factors for biliary infection. Continuous data were compared using the nonparametric Mann–Whitney U test. Frequency differences were compared using the chi-squared test or Fisher’s exact test where appropriate. The covariates that showed a high level of significance in the univariate analysis were analyzed further in a multivariate analysis using the binary logistic regression method. Actuarial retransplantation-free survival was estimated using a Kaplan–Meier product limit estimator. Differences between the actuarial estimates were tested using the log rank test. Cox regression was used to calculate the hazard ratio. Differences were considered significant when P < 0.05. Statistical analyses were performed using PASW Statistics 17.0 (SPSS, Chicago, Illinois, USA).
Table 2 Endoscopic findings in 213 patients at baseline (index endoscopic retrograde cholangioscopy [ERC] for biliary complications following orthotopic liver transplant.
n Indication for primary ERC Unexplained progressive cholestasis 1
91
42.7
Suspected stricture 1
72
33.8
Suspected leakage 1
38
17.8
Suspected gallstones 1
9
4.2
Cholangitis
3
1.4
49
23.0
Anastomotic stricture
52
24.4
Nonanastomotic stricture
39
18.3
Leakage
24
11.3
Previous endoscopic treatment (EST, balloon dilation, plastic stent, percutaneous drainage) Findings at primary ERC 2
Gallstones
10
4.7
Purulent cholangitis 3
12
5.6
115
53.9
Endoscopic treatment (balloon dilation, plastic stent, metal stent, stone removal, EST alone)
ERC, endoscopic retrograde cholangiography; EST, endoscopic sphincterotomy; 1 With or without signs of cholangitis 2 Where combined lesions were present, the clinically more prominent one was used 3 Purulent cholangitis was observed with or without other pathological findings
Table 3 Success of endoscopic treatment. Therapeutic procedures were carried out at the index ERC in 115 of the 213 patients with biliary complications after orthotopic liver transplant.
n/n
%
Sustained clinical success
60/115
52.2
Assisted clinical success
21/115
18.3
Combined (sustained or assisted) clinical success
81/115
70.4
Therapeutic procedure at first ERC (n = 115)
Success not evaluable Treatment failure
Results !
Endoscopic findings and treatment after liver transplantation Overall, 213 patients who had undergone liver transplantation were included in this prospective study. The underlying liver diseases had been chronic viral hepatitis (n = 63), alcoholic liver disease (n = 70), hepatocellular carcinoma (n = 41), or other indications (n = 39). The median time interval between transplantation and the occurrence of the biliary complication requiring the index ERC was 3.0 months (0 – 252 months). Indications for performing ERC included suspected stricture (n = 72), leakage (n = 38) or bile duct stones (n = 9). Out of the 213 patients, 21 presented with clinical signs of cholangitis; laboratory parameters of inflammation for the 213 patients were as follows: WBC mean (SD) 7.6 (5.0) /nL, CRP 31.0 (42.9) mg/L (upper limit of normal 5.0 mg/L). Three patients presented with clinical signs of severe cholangitis or sepsis, but in a substantial proportion of patients with progressive cholestasis (n = 91) neither ultrasound, magnetic resonance imaging (MRI), computed tomography (CT) scan, nor histology was able to provide a reliable diagnosis. Therefore in these patients ERC was performed primarily with a diagnostic intention. This led to definite diagnosis in 36 % of these patients. In 23 % (49/213) of patients a previous endoscopic biliary intervention was documented (endoscopic sphincterotomy [EST] 11.3 %, balloon dilation 6.6 %, plastic stent placement 3.8 %, other
%
Recurrent or newly developed biliary lesion (among all patients, n = 213)
7/115
6.1
27/115
23.5
21/213
9.8
ERC, endoscopic retrograde cholangioscopy
1.4 %). This intervention had been either performed before OLT or at a different endoscopy unit. During the ERC, the most common finding was anastomotic stricture, followed by nonanastomotic or diffuse intrahepatic strictures, leakage or gallstones. In 115 patients (53.9 %) one or more therapeutic endoscopic pro" Table 2). The sustained clinical succedures were performed (● cess rate in these patients was 52 % (60/115) and the assisted clinical success rate 18 % (21/115). Thus, an overall clinical success after the endoscopic interventions was achieved in 70 % of patients. Treatment failure occurred in 23.5 % (27/115). Recurrence of the biliary complication or a newly developed biliary lesion " Taduring follow up was observed in 10 % (21/213) of patients (● ble 3).
Detection rates for bacteria and fungi in bile Among all the ERC examinations of the 213 patients at inclusion into the prospective study, bile cultures were not performed in 33 cases because of technical or organizational failure. In total, 102/180 (57 %) of patients had Gram-positive and 44/180 " Table 4). (24 %) had Gram-negative bacteria detectable in bile (● Enterococcus species were predominant (72/180 samples; 40 %). Escherichia. coli and Klebsiella spp. were detected in 10 % (18/180) and 4 % (7 /180) of the samples, respectively. Alpha- and beta-he-
Gotthardt Daniel N et al. Bacteriobilia and fungibilia and endoscopic treatment of complications after liver transplantation … Endoscopy 2013; 45: 890–896
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892
Table 4 Microbiological isolates from bile specimens at first endoscopic retrograde cholangiography (ERC).
Organism
n
%1
Bacteria At least one Gram-positive:
102
56.7
Enterococcus spp.
72
40.0
Streptococci, alpha- and beta-hemolytic
37
20.6
Staphylococcus spp.
23
12.8
Other
9
5.0
44
24.4
Escherichia coli
18
10.0
Klebsiella spp.
7
3.9
Enterobacter cloacae
3
1.7
Neisseria spp.
3
1.7
Citrobacter spp.
2
1.1
Pseudomonas aeruginosa
2
1.1
18
10.0
1
0.6
At least one Gram-negative:
Other Multiresistant bacteria Vancomycin-resistant enterococci (VRE) Multiresistant Enterococcus faecium, not VRE
12
6.7
Extended spectrum beta lactamase (ESBL)
2
1.1
Other multiresistant strains
7
3.9
Fungi Candida albicans
1
24
15.6
C. tropicalis
2
1.3
C. glabrata
7
4.5
Candida spp.
3
1.9
Sterile
54
(30.0)
No information available
33
(15.5)
Percentage is based on either the total number of ERCs for which microbiological analysis was available (n = 180) or the total number of ERCs where bile was in addition tested for fungi (n = 154)
Table 5
Microbiological spectrum of bile samples.
%1
n
1
Sterile
54
30 %
Low grade pathogens
57
32 %
Enteric
85
47 %
Candida
28
18 %
Any multiresistant strain
21
12 %
As percentage of ERCs for which microbiological information was available (154 for Candida; 180 for the rest).
Bile sample microbiology
Laboratory signs of cholangitis
molytic streptococci were detected in 21 % of the samples and coagulase-negative staphylococci in 13 %. The Gram-negative species were Enterobacter cloacae (2 %), Citrobacter spp. (1 %), and Pseudomonas aeruginosa (1 %). Multiresistant bacteria included the following: 1 % vancomycin-resistant Enterococcus (VRE) faecalis, 1 % E. coli with extended spectrum beta-lactamase (ESBL) resistance, and 7 % Enterococcus faecium strains that were only sensitive to " Table 4). vancomycin, linezolid, or other reserve antibiotics (● We also analyzed the bile samples for fungi. A total of 16 % showed Candida albicans; however, other Candida species were " Table 4 and ● " Table 5). Some Candida detected at low rates (● species, including C. glabrata and C. tropicalis, were detected at low frequencies, while still other fungal organisms, such as Aspergillus fumigatus, were not detected. Summarizing the results of analysis resulted in the following: 54/ 180 (30 %) sterile bile cultures; 57/180 (32 %) samples with low grade pathogens; 85/180 (47 %) with at least one enteric bacterial strain; 28/180 (18 %) samples with Candida. At least one multiresistant strain was detected in 21 /180 (12 %) of the samples. These " Table 5. data are summarized in ●
Risk factors for detection of microbiological isolates according to univariate and multivariate analyses For analysis of bacterial isolates we included 180/213 patients in the analysis, while for patients with fungibilia we included 154/ 213 patients, since in 33 patients microbiological data were not available and for 26 patients data on bacteribilia, but not fungibilia were available. Neither bacteriobilia arising from enteric bacteria nor detection of Candida was associated with increased serum levels of CRP " Table 6). WBC was not associated with the presence of bacter(● ia or fungi. The clinical signs of cholangitis were recorded before ERC treatment and were much more frequent in the groups with enteric bacteribilia and fungibilia (P < 0.05 for all comparisons). There was no association with either of the low grade pathogen groups (data not shown). As with the clinical signs of cholangitis, patient age and previous endoscopic intervention were associated with the detection of " Table 7). Lower levels found in liver function enteric bacteria (● test (LFT) results were significantly associated with the detection
Clinical signs of inflammation Values are n (%)
WBC, /nL
CRP, mg/L
– ve
+ ve
Sterile
7.8 (4.8)
27.7 (43.1)
51 (96 %)
2 (4 %)
Not sterile
7.2 (4.7)
32.4 (45.1)
111 (87 %)
16 (13 %)
Univariate analysis
P = 0.135
P = 0.583
P = 0.101
Multivariate analysis 1
—
—
—
Enteric– ve
7.5 (4.4)
27.3 (42.2)
91 (96 %)
3 (4 %)
Enteric + ve
7.1 (5.1)
35.1 (47.3)
71 (84 %)
15 (16 %)
Univariate analysis
P = 0.361
P = 0.313
P = 0.003
Multivariate analysis 1
—
—
P = 0.051
Candida – ve
7.4 (5.0)
31.2 (46.7)
119 (94 %)
7 (6 %)
Candida + ve
7.4 (4.5)
32.8 (45.9)
22 (79 %)
6 (21 %)
Univariate analysis
P = 0.868
P = 0.434
Multiresistant – ve
7.5 (4.8)
29.9 (44.4)
134 (90 %)
15 (10 %)
Multiresistant + ve
7.9 (5.9)
36.7 (37.5)
18 (86 %)
Univariate analysis
P = 0.799
P = 0.157
Table 6 Clinical and laboratory signs of cholangitis. Univariate and multivariate analysis. Parameter values are means (standard deviation [SD]), with units as shown.
P = 0.006 3 (14 %) P = 0.470
WBC, white blood cell count; CRP, C-reactive protein; – ve, absent; + ve, present WBC and CRP were analyzed using the Mann – Whitney U test. Significance of differences was calculated using the chi-squared or Fisher’s exact test where applicable. 1 Parameters with P < 0.1 in univariate analysis were included in a multivariate analysis. The P values from this analysis are reported here.
Gotthardt Daniel N et al. Bacteriobilia and fungibilia and endoscopic treatment of complications after liver transplantation … Endoscopy 2013; 45: 890–896
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Table 7 Patient age, liver function tests and previous endoscopic intervention associated with detection of bacteria and fungi at first ERC. Parameter values are means (SD), with units as shown.
Bile sample
Age, years
AST, IU/L
ALT, IU/L
ALP, IU/L
gGT, IU/L
microbiology
Bilirubin,
No previous inter-
Previous endoscopic
mg/dL
vention, n(%)
intervention, n (%)
Not sterile
54.2 (10.7)
101 (137)
140 (211)
358 (313)
628 (601)
3.1 (3.8)
87 (68 %)
Sterile
51.9 (9.1)
117 (96)
203 (235)
482 (395)
854 (684)
3.7 (3.5)
47 (89 %)
40 (32 %)
Univariate analysis
P = 0.034
P = 0.007
P = 0.001
P = 0.019
P = 0.024
P = 0.058
Multivariate analysis 1
P = 0.684
P = 0.395
P = 0.158
P = 0.175
P = 0.890
P = 0.782
Enteric– ve
52.4 (9.3)
115 (108)
183 (217)
428 (380)
786 (645)
3.3 (3.3)
83 (87 %)
12 (13 %)
Enteric + ve
55.1 (11.1)
97 (146)
132 (224)
345 (285)
582 (583)
3.3 (4.2)
51 (60 %)
34 (40 %)
Univariate analysis
P = 0.005
P = 0.004
P = 0.001
P = 0.073
P = 0.012
P = 0.088
Multivariate analysis
P = 0.563
P = 0.675
P = 0.202
P = 0.415
P = 0.625
P = 0.444
Candida – ve
53.8 (10.6)
99 (112)
142 (181)
395 (335)
709 (621)
3.3 (3.8)
99 (79 %)
27 (21 %)
Candida + ve
52.4 (10.0)
153 (194)
215 (317)
444 (412)
616 (693)
3.5 (4.0)
18 (64 %)
10 (36 %)
Univariate analysis
P = 0.529
P = 0.823
P = 0.908
P = 0.745
P = 0.211
P = 0.914
Multiresistant – ve
53.8 (10.5)
103 (104)
152 (193)
402 (352)
688 (620)
3.4 (3.9)
111 (75 %)
Multiresistant + ve
52.0 (10.4)
150 (246)
238 (384)
381 (287)
837 (711)
3.5 (3.7)
17 (81 %)
Univariate analysis
P = 0.649
P = 0.370
P = 0.771
P = 0.966
P = 0.354
P = 0.837
6 (11 %) P = 0.005 P = 0.007
P < 0.001 P = 0.001
P = 0.110 38 (25 %) 4 (19 %) P = 0.601
ERC, endoscopic retrograde cholangioscopy; SD, standard deviation; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; gGT, gamma-glutamyltransferase. The Mann – Whitney U test was used to analyze for laboratory values; the chi-squared test was used to compare intervention. 1 Parameters with P < 0.1 in univariate analysis were included in a multivariate analysis. The P values from this analysis are reported here.
" Taof enteric bacteria and the detection of any isolate in bile (● ble 7). Parameters that were associated with a P value < 0.1 were all analyzed using a logistic regression model. For detection of sterile bile and for the detection of enteric bacteria in bile only a previous intervention remained significant (P < 0.01). We assessed whether the presence of bacteribilia and/or fungibilia was associated with endoscopic findings in patients after liver transplantation. There was no significant association with the frequency of endoscopic findings nor with the frequency of sustained endoscopic therapeutic success with the detection rate of microbiological isolates (P > 0.05, chi-square test; data not shown).
Association of bacteriobilia and fungibilia with retransplantation free survival We assessed whether the presence of bacteriobilia and/or fungibilia was associated with outcome in patients undergoing endoscopic treatment after liver transplantation. Patients with detection of enteric bacteria at first ERC had no difference in regard to retransplantation-free survival compared with patients without enteric bacteria, if endoscopic treatment success was not considered. In patients who were successfully treated by endoscopic intervention, the actuarial retransplantation-free survival was significantly shorter when enteric bacteria were detected: in those positive for enteric bacteria it was 51.8 months (95 % confidence interval [CI] 42.9 – 60.6), vs. 62.9 months (95 %CI 59.1 – 66.7) in those negative for enteric bacteria, " Fig. 1 a), with a hazard ratio of 7.620. with P = 0.025 (● Fungibilia was associated with significantly shorter actuarial retransplantation-free survival, independently of the success of endoscopic treatment. Patients with detection of Candida had a mean actuarial retransplantation-free survival of 35.1 months (95 %CI 23.5 – 46.7) whereas those without Candida had a mean " Fig. 1 b), with a hazard raof 53.1 months (95 %CI 48.0 – 58.2) (● tio of 2.590. When patients with a previous endoscopic intervention were excluded the results were as follows. Patients who were successfully treated, showed an actuarial retransplantation-free survival of
62.4 months (95 %CI 57.6 – 67.2) if no enteric bacteria were detected vs. 51.4 months (95 %CI 39.9 – 62.8) if enteric bacteria were present (P = 0.057). Patients without a previous intervention and without Candida had a mean actuarial retransplantation-free survival after first ERC of 50.4 months (95 %CI 44.4 – 56.4); whereas patients with Candida showed a mean of 27.5 months (95 %CI 16.3 – 38.7) (P = 0.049.
Discussion !
Cholangitis is a serious complication of biliary obstruction that requires biliary drainage and antibiotic treatment [10 – 12, 21]. Following transplantation, infectious complications are a major cause of morbidity and mortality [22]. Immunosuppression is one factor contributing to this, but in addition, in patients who have had liver transplantation the biliary tract is impaired in its physiological function to a varying extent. Therefore cholangitis can occur more easily. This leads to biliary tract infection as a major complication in these patients. Data on frequencies of bacteriobilia and cholangitis in patients after OLT are scarce, and were regularly obtained from patients with a T-tube drain in place during the first days after transplantation. Fungal species are emerging as pathogens that are detected frequently in the setting of healthcare-associated biliary infection. In agreement with previous reports [23, 24] we detected Candida species, mainly C. albicans in bile samples. We were able to confirm the high frequency of bacteriobilia and fungibilia that has been reported by Millonig et al. in a smaller previous study [19]. In our cohort, only 30 % of samples were sterile. These numbers are noteworthy, since bile is normally considered to be sterile. It is of interest that even at first ERC almost 50 % of samples showed enteric bacteria and 18 % showed Candida. One of the main findings of this study is the clear benefit of routine bile sampling during ERCs. Bile cultures can be used to identify bacterial strains and, more importantly, if performed routinely reveal a patient’s susceptibility profile. Blood cultures are far less sensitive for detecting this information and the inclusion of such data from bile sampling can lead to changes in the anti-
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1.0 enteric –ve 0.8
Cumulative survival
enteric +ve 0.6 P < 0.05 0.4
0.2
enterio-negative enterio-positive censored censored
0.0 0 a
20 40 Time from first ERC, months
60
1.0
Candida –ve
Cumulative survival
0.8
0.6 Candida +ve 0.4 P < 0.05 0.2
Candida-negative Candida-positive censored censored
0.0 0 b
20 40 Time from first ERC, months
60
Fig. 1 Bacteriobilia and fungibilia in liver transplantation patients: association with survival without further transplantation. The microbiological assessment was done at a first endoscopic retrograde cholangioscopy (ERC) performed because of biliary complications after orthotopic liver transplantation (OLT). All time periods are from the first ERC of the study. a Among patients who were successfully treated by endoscopic intervention, those with no enteric bacteria in their bile showed a significantly better actuarial survival free of retransplantation, with a mean 62.9 months (95 % confidence interval [CI] 59.1 – 66.7) vs. those with enteric bacteriobilia, with a mean 51.8 months (95 %CI 42.9 – 60.6); P = 0.025. b Patients without Candida present in their bile had a higher retransplantation-free survival, with a mean of 53.1 months (95 %CI 48.0 – 58.2) vs. patients with Candida with a mean of 35.1 months (95 %CI 23.5 – 46.7); P = 0.019. This finding was independent of success of endoscopic treatment.
biotic treatment [18]. This is especially important in patients who have had liver transplantation, because such patients are regularly hospitalized and treated for infectious complications of different kinds. Since we could not identify clear risk factors for the detection of multiresistant strains, we argue for the acquisition of bile samples during each ERC. Although bile cultures are more sensitive, blood cultures are still standard in patients with signs of infection. Usually acquisition of bile is possible without any difficulty. In cases when aspiration is cumbersome, the endoscopist needs to weigh the potential benefit of bile culture ver-
sus the potentially higher risk of the procedure and contamination of the sample because of prolonged acquisition time. In our cohort the predominance of Enterococcus spp. was even more obvious than had been anticipated, with 40 % of all samples being positive for any kind of Enterococci. This is in line with other data relating to intra-abdominal infection post-OLT [25] but it is higher compared with other reports from patients with obstructive jaundice [14, 15, 26], and is of special importance because Enterococcus spp. have been shown to be largely resistant to fluoroquinolones, especially ciprofloxacin [27]. Ciprofloxacin has been widely used as peri-interventional prophylaxis in ERC patients and in treatment of cholangitis. Our data would argue in favor of a broad spectrum penicillin or, in cases of any known multiresistant bacterial strain in bile and cholangitis, for even broader antimicrobial therapy such as carbapenems. On the basis of our data we cannot give a recommendation on the duration of antibiotic treatment. We found different Candida species in our analysis. No other fungal organism, e. g. Aspergillus spp., was identified. Therefore treatment with fluconazole should be sufficient, but the optimal duration of treatment has to be determined. Usually we treat for 3 – 4 weeks which is longer than treatment for bacteria. The clinical relevance of bacteriobilia and fungibilia has been questioned. We have demonstrated their clinical relevance by the significant association between clinical signs of cholangitis with bacteriobilia/fungibilia although numerous patients had bacteriobilia/fungibilia and no clinical sign of cholangitis. This might either reflect the impaired immunological response in the immunosuppressed patient, a subclinical course of cholangitis, or simply the altered biliary microenvironment of these patients after OLT. Although all patients received antibiotic treatment, eradication of the bacteriobilia was not achieved. So in post-OLT patients, the biliary tract can no longer be considered to be a sterile environment. This altered biliary microenvironment might be further challenged by changes in the biliary lipid composition leading to nonanastomotic stricture and graft failure, resembling biliary cirrhosis caused by reduced phospholipid content [28, 29]. We could not find a significant association with the endoscopic findings nor with the success rate of the endoscopic treatment. In a considerable proportion of patients (43 %) ERC was performed with a diagnostic purpose, because histological examination as well as imaging procedures were not able to clarify the progressive cholestasis. As a definitive diagnosis was thereby established in more than one third of patients, this approach appears appropriate. The influence on survival has not been addressed in former studies on OLT patients, but has revealed significant results in patients with PSC [30, 31]. The detection of Candida was associated with a markedly shorter retransplantation-free survival. Whether this effect is due to a chronic, damaging infection of the biliary tract or whether biliary tract candidiasis is only another marker of disease has yet to be determined. Furthermore we were able to show that the initial absence of enteric bacteria is associated with longer retransplantation-free survival in patients who respond to endoscopic therapy. This is noteworthy, because it shows that the altered microbiological environment of the biliary tract after liver transplantation contributes to outcome in these patients. We also confirmed the excellent results of endoscopic therapy in patients with biliary tract complications after liver transplantation [32 – 34]. Two points have to be kept in mind when interpreting our data. The first concerns the potential cross-contamination. As all routi-
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nely performed cultures of endoscopes, involved surfaces, and worktops were negative, cross-contamination from reprocessing and storage of endoscopes could be excluded; however crosscontamination during the upper gastrointestinal passage of the endoscope may have influenced the results. The other point is that many of our patients had received antibiotic treatment before bile sampling was performed. Since the exact timing of application of the antibiotics and bile sampling could not be retrieved, potential antimicrobiological activity in the bile might influence the microbiological assessment. In conclusion, in post-liver transplantation patients, bacteriobilia and fungibilia can frequently be detected by routine microbiological sampling, thereby allowing optimized, strain-specific antibiotic treatment. Both have clinical significance and we also identified significant risk factors for the detection of bacteria and Candida. Since we could not identify clear risk factors for the detection of multiresistant strains, we argue for acquisition of bile samples during each ERC. This would enable the physician to administer targeted strain-specific treatment. For the first time, we assessed survival in these patients on the basis of biliary tract microbiology and depending on their endoscopic treatment and the success rate of endoscopic treatment. We demonstrated a shorter retransplantation-free survival in patients with Candida or enteric bacteria present in their bile. The influence of this microbiologically altered biliary environment on chronic graft failure and its treatment options warrants further study. Competing interests: None
Acknowledgments !
We thank Tom Bruckner, Department of Medical Biometry and Informatics, for critical review of the statistic analyses. D.G. and K.H.W. were supported by the post-doctoral program of the Medical Faculty of the University of Heidelberg. D.G. was also supported by a grant from the Deutsche Forschungsgemeinschaft.
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